Characterization and Development of Strategies for Altering Protein Expression in JSL1 Cells

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Title: Characterization and Development of Strategies for Altering Protein Expression in JSL1 Cells
Author: Senitko, Annette Nelson
Abstract: Alternative splicing is a common mechanism for regulating gene expression in eukaryotic cells . This process of differentially including or excluding variable exons provides a means for increasing proteome complexity . Alternative gene splicing occurs in a cell specific manner and may be influenced by changes in the extracellular environment . Despite the importance of this method for regulating gene expression , little is known about the factors involved in regulating its function . The T cell tyrosine phosphatase CD45 provides a valuable model for investigating the factors involved in regulating alternative splicing . The CD45 gene contains three variable exons whose splicing is regulated in response to T cell activation . Studies of this gene have revealed the presence of an exonic silencer sequence within variable exon 4 that is capable of influencing exon skipping under both resting and stimulated conditions . Biochemical assays have shown that the regulatory protein hnRNP L binds to this silencer sequence and results in basal exon repression during resting conditions and undergoes modifications which further influence exon skipping upon stimulation . Furthermore , in vitro assays indicate that upon stimulation , an additional regulatory protein , PSF , binds to the regulatory complex associated with the silencer sequence . Although these studies have provided novel information regarding the regulation of splicing , biochemical assays are unable to fully mimic the signaling pathways inside a cell , thus creating a need for a cell culture system . A Jurkat derived cell line , JSL1 cells , has been identified as being able to recapitulate the signal induced alternative splicing of the CD45 gene as seen in primary human T cells . This cell line presents a cell based system for evaluating the factors involved in splicing . However , in order to conduct in vivo experiments one must be able to modify protein expression . JSL1 cells present limitations due to difficulties in being able to alter protein expression . A strong promoter , EF1 -alpha , has been employed to drive the expression of candidate proteins in JSL1 cells . Transient transfections and stable cell lines expressing cDNAs driven by this promoter have shown little if any overexpression of candidate proteins normally expressed at high levels within the cell ; however , significant overexpression has been achieved with the transfection of at least one protein that exists at a lower concentration . Initial experiments indicate that stably expressed flag -tagged proteins , driven by the EF1 -alpha promoter , may be easily purified from JSL1 cells during resting and stimulated conditions and analyzed . Such data suggests that this promoter may afford more flexibility in altering and analyzing protein expression in JSL1 cells , thereby facilitating the investigation of signaling pathways involved in regulating alternative splicing . Furthermore , strategies for regulating protein expression , through the use of a Tet -suppressor system , are in initial stages of being developed and hold the potential for providing an additional tool for evaluating the factors involved in regulating alternative splicing .
URI: http : / /hdl .handle .net /2152 .5 /241
Date: 2007-08-08

Citation

Characterization and Development of Strategies for Altering Protein Expression in JSL1 Cells. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /241 .

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